The use of retroviral vectors for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani et al., Human Gene Therapy 5:19-28 (1994)). However most of these protocols require that the infection of target cells with the retroviral vector carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg et al., Hum. Gene Ther. 3:75-90 (1992); for a review see Anderson, W. F., Science 256:808-813 (1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g. lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of retroviral vector producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed. Virus will be produced, possibly from an implantation of virus producing cells, and there will be no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk.
Retroviral vector systems consist of two components (FIG. 1):
1) the retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes and marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.The second component is:2) a cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with a second plasmid carrying the genes enabling the modified retroviral vector to be packaged.
To generate the packaged vector, the vector plasmid is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic and marker genes is transcribed from the vector plasmid and packaged into the modified retroviral particles (recombinant viral particles). This recombinant virus is then used to infect target cells in which the vector genome and any carried marker or therapeutic genes becomes integrated into the target cell's DNA. A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells. However the DNA of the vector carrying the therapeutic and marker genes is integrated in the cell's DNA and can now be expressed in the infected cell.
The essentially random integration of the proviral form of the retroviral genome into the genome of the infected cell (Varmus, Science 240:1427-1435 (1988)) led to the identification of a number of cellular proto-oncogenes by virtue of their insertional activation (Varmus, Science 240:1427-1435 (1988); van Lohuizen and Berns, Biochim. Biophys. Acta, 1032:213-235 (1990)). The possibility that a similar mechanism may cause cancers in patients treated with retroviral vectors carrying therapeutic genes intended to treat other pre-existent medical conditions has posed a recurring ethical problem. Most researchers would agree that the probability of a replication defective retroviral vector, such as all those currently used, integrating into or near a cellular gene involving in controlling cell proliferation is vanishingly small. However it is generally also assumed that the explosive expansion of a population of replication competent retrovirus from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. However it has been well documented that recombination events between components of the retroviral vector system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (reviewed in Salmons and Gunzburg, Human Gene Therapy 4:129-141 (1993)).
A further consideration when considering the use of in vivo gene therapy, both from a safety stand point and from a purely practical stand point, is the targeting of retroviral vectors. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumor cells. Ablation of other, nontarget cells would obviously be very undesirable.
A number of retroviral vector systems have been previously describes that should allow targeting of the carried therapeutic genes (Salmons and Gunzburg, Human Gene Therapy 4:129-141 (1993)). Most of these approaches involve either limiting the infection event to predefined cell types or using heterologous promoters to direct expression of linked heterologous therapeutic or marker genes to specific cell types. Heterologous promoters are used which should drive expression of linked genes only in the cell type in which this promoter is normally active. These promoters have previously been inserted, in combination with the marker or therapeutic gene, in the body of the retroviral vectors, in place of the gag, pol or env genes.
The retroviral Long Terminal Repeat (LTR) flanking these genes carries the retroviral promoter, which is generally non-specific in that it can drive expression in many different cell types (Majors, Curr. Tops. In Micro. Immunol. 157:49-92 (1990)). Promoter interference between the LTR promoter, and heterologous internal promoters, such as the tissue specific promoters described above has been reported. Additionally, it is known that retroviral LTRs harbor strong enhancers that can, either independently, or in conjunction with the retroviral promoter, influence expression of cellular genes near the site of integration of the retrovirus. This mechanism has been shown to contribute to tumorigenicity in animals (van Lohuizen and Berns Biochim. Biophys. Acta, 1032:213-235 (1990)). These two observations have encouraged the development of Self-Inactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (PCT WO94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (PCT WO89/11539). However, in both these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the marker/therapeutic gene.
The previously described SIN vector mentioned above carrying a deleted 3′LTR(PCT WO94/29437) utilize in addition a strong heterologous promoter such as that of Cytomegalovirus (CMV), instead of the retroviral 5′ LTR promoter (U3-free 5′LTR) to drive expression of the vector construct in the packaging cell line. A heterologous polyadenylation signal is also included in the 3′LTR (PCT WO94/29437).